The present invention relates generally to analytical devices for performing assays and more particularly to an analytical device that includes a centrifugal force actuated valve to control fluid and a method of utilizing the analytical device to perform an assay.
There are many analytical methods that require one or more reactions or analytical steps to determine a final answer. Examples are heterogeneous immunochemistry reactions, hybridization of DNA to DNA, and hybridization of DNA to RNA. In order to determine the concentration or presence of an analyte, such analytical methods require multiple, serial reactions either with or without washing steps or a single reaction with a washing step. Other assays, such as clinical chemistry assays, often require precise dilutions prior to mixing with other chemicals.
It would be desirable to reduce the cost of conducting the reactions in an assay by automating the reaction steps rather than using expensive manual labor to perform the steps. Additionally, it would be advantageous to use centrifugal force in the automation process to minimize variations due to surface tension and capillary action and to move and control fluid.
Centrifugally driven analytical devices have employed several methods for controlling fluid movement, such as differential flow, stop junctions, siphons, and complex, two-axis mechanisms.
Differential flow allows fluid to enter a chamber quickly but exit slowly. As the fluid enters and exits the chamber there is a finite residence time in which most of the fluid can be manipulated. However, the disadvantage of differential flow is that the entire volume of fluid is never completely controlled.
Stop junctions employ the pressure created by a capillary to stand off fluid flow until the centrifugal force generated by rotation overcomes capillary back pressure. Stop junctions are sensitive to the exact geometry and surface properties of the junction and to fluid properties of the sample.
Siphons allow fluid movement into a chamber under the action of centrifugal force and prevent the fluid from exiting the chamber until the siphon is primed or the siphon level is high enough. The disadvantage with siphons is that some of the fluid is lost in the entrance of the siphon and that great care must be taken to prevent the siphon from priming prematurely or from losing prime prematurely.
Two-axis mechanisms provide better fluid control, but at substantial instrumentation cost, complexity, and size. Two-axis mechanisms are typically mounted on a large turntable. The turntable has two positions, each position having a local center of rotation.
U.S. Pat. No. 5,171,533 to Fine et al. discloses single use centrifugal valves for performing a biological assay. Sealant materials are used to provide a counteracting force to the centrifugal force of rotation. The sealants are designed to yield at predetermined levels of centrifugal force. The valves in the Fine et al. patent require that the fluid move through different chambers.
From the foregoing it will be apparent that there is a need for a simple, easy to automate, and economical means for performing an assay or a reaction. Further, it will be apparent that a disposable analytical device is desirable for preventing contamination resulting from repeated use of the same analytical device.
The invention provides an analytical device incorporating a centrifugal force-activated valve for controlling reactions and for moving fluid within the analytical device. The centrifugal force-activated valve allows for several fluids to be passed through the analytical device and allows the analytical device to be used for multiple serial reactions. The centrifugal force-activated valve provides simple and reliable control over fluid movement and can be tuned to open or close over a wide range of rotational speeds.
An analytical device embodying the invention is economical, easy to use, injection moldable from a variety of plastics, readily adaptable to automation, capable of repeated reaction, mixing, and washing steps, disposable to prevent contamination, and may be transparent to allow an assay by optical detection means.
In a preferred embodiment of an analytical device according to the present invention, a housing enclosure adapted for rotation about an axis includes an assay chamber, a fluid discharge port in fluid communication with the assay chamber, means for introducing fluid into the assay chamber, and a centrifugal force-activated valve in fluid communication with the fluid discharge port. Preferably, the fluid introducing means is self sealing. Fluid entering the valve through the fluid discharge port may exit the valve through a fluid drain. The fluid drain is in fluid communication with the valve and an exterior portion of the housing enclosure. Chemical, biological, or biochemical fluids may be introduced into the assay chamber.
Additionally, an active surface can be positioned on an interior surface of the assay chamber. The active surface may contain biomolecular probes. The biomolecular probes can be DNA, DNA fragments, RNA, RNA fragments, reagents, protein, protein fragments, lipids, and lipid fragments. Additionally, the active surface can be disposed on a structure positioned on an interior surface of the assay chamber. For example, the structure can be in the shape of balls or beads. The structure can be attached to an interior surface of the assay chamber or can be loosely contained in the assay chamber. Typically, the active surface is arranged in an array pattern positioned on a surface of the assay chamber.
An analytical substrate can be positioned in an opening in the housing enclosure. The active surface can be disposed on an interior surface of the analytical substrate and in opposing relation to the assay chamber.
The analytical substrate can be made transparent to optical detection means external to the housing enclosure. A transparent analytical substrate allows for an assay of the active surface by scanning the active surface through the analytical substrate using the optical detection means.
The analytical device may also include a reservoir chamber disposed radially outward from the centrifugal valve and in fluid communication with the valve. Fluid exiting the centrifugal valve is stored in the reservoir chamber. Optionally, fluid contained in the reservoir chamber can be extracted through fluid extracting means in fluid communication with the reservoir chamber. Preferably, the fluid extraction means is self sealing.
In another embodiment, the housing enclosure includes a portion defining a valve chamber for containing the centrifugal force-activated valve. A fluid discharge port is positioned at a radially inward end of the valve chamber and is in fluid communication with the valve chamber and the assay chamber. A valve seat is disposed in the valve chamber and is disposed radially outward from the fluid discharge port. The valve seat is in fluid communication with the fluid discharge port and the valve chamber. An actuator is movably disposed in the valve chamber and is biased to a stationary position relative to the valve seat by a counter acting force provided by bias means positioned in the valve chamber and disposed radially outward from the actuator. The actuator moves radially outward from the stationary position to an actuated position when the housing enclosure is rotated and the centrifugal force acting on the actuator exceeds the counteracting force of the bias means. When the centrifugal force drops below the counteracting force, the actuator moves radially inward from the actuated position to the stationary position.
A method according to the present invention includes injecting a sample into the assay chamber through the fluid introducing means. The analytical device is then spun at a first rotational speed lower than that required to actuate the centrifugal force-activated valve. The first rotational speed needs to be sufficient to distribute the sample across the active surface. The sample is allowed to react with the active surface. At the conclusion of the reaction period, the analytical device is spun at a second rotational speed to actuate the centrifugal force-activated valve and empty the sample from the assay chamber. Spinning at the second rotational speed is stopped when the assay chamber is empty. Wash fluids are injected into the assay chamber and the analytical device is spun at the first rotational speed to distribute the wash fluids. The analytical device is then spun at the second rotational speed to empty the wash fluids. The washing step can be repeated as necessary to effect complete washing of the assay chamber and the active surface. After the washing step, the active surface can be scanned to detect the presence of an analyte.
Other aspects and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, illustrating by way of example the principles of the invention.